PROJECT SUMMARY/ABSTRACT Coenzyme A (CoA) is an essential cofactor required for hundreds of metabolic processes. Because it is such a critical cofactor, CoA levels are tightly regulated. In diabetic mice, an abnormally high concentration of CoA in the liver drives excess glucose production and contributes to the hyperglycemia of these animals. This phenotype is associated with constitutively low CoA degradation, a process that is emerging as a potentially important mechanism for CoA regulation. Nudt7 and Nudt19 are two mammalian peroxisomal enzymes with CoA-degrading activity, which are highly expressed in the liver and kidney, respectively. Limited information is available on the biochemistry of Nudt7 and Nudt19; the structural basis for their distinct features and the extent to which Nudt7 and Nudt19 contribute to maintaining homeostatic CoA levels in vivo are currently unknown. The proposed research will use a combination of techniques including mutagenesis, molecular modeling, enzymatic assays on purified proteins, and targeted metabolite analysis in whole tissue homogenates and subcellular fractions to: 1) characterize the biochemical, structural, and regulatory properties of Nudt7 and Nudt19 and 2) determine the effects that genetic manipulations of Nudt7 have on subcellular CoA levels in mouse liver and on glucose metabolism. This research will establish the importance of degradation in the regulation of CoA and the potential of targeting this process to control and correct dysregulated metabolism in the liver and kidneys.